Primary culture experiment of neonatal rat cardiomyocytes
Primary culture experiment of neonatal rat cardiomyocytes
Primary culture of neonatal rat cardiomyocytes can be applied to (1) cell preservation, (2) for cell morphology study, and (3) for clinical research.
Operation method
Primary culture technique
Principle
Cardiomyocytes from rats were removed from the organism, treated with trypsin, chelating agent (commonly used EDTA), dispersed into single cells, and cultured in a suitable medium to allow the cells to survive, grow, and multiply.
Materials and Instruments
SD suckling mouse Move I. Preparation of experimental materials Caveat Mammalian rat cardiomyocytes are primary growth cells and the culture process is cumbersome. There are various concentrations of trypsin digestion methods in the literature, 0.06% concentration of trypsin is less damaging to the cells, but this concentration of digestion takes a longer time. Adopt the method of repeated low concentration digestion for many times, after digesting some each time, extract the supernatant with a pipette, and centrifuge the result that is the cardiomyocytes. Taking advantage of the difference in wall-application time between cardiomyocytes and fibroblasts, differential wall-application was used for 1.5 h to remove fibroblasts sufficiently and achieve the purpose of purification. Fibroblasts have a pike-shaped or irregularly triangular cytosol with an ovoid nucleus in the center, protruding cytoplasm, radial growth, and proliferation without pulsation, which is good to distinguish from cardiomyocytes. Digestion and blowing must not be excessive, is the most important one to ensure the viability of cardiomyocytes. While the inoculum density (generally not less than105/ml), will also affect the beating of cardiomyocytes and synchronized beating. Common Problems I. Experimental discussion Mammalian rat cardiomyocytes belong to primary growth cells, and the culture process is cumbersome. There are various concentrations of trypsin digestion methods in the literature, 0.06% concentration of trypsin is less damaging to the cells, but the time required for digestion at this concentration is longer. Adopt the method of repeated low concentration digestion for many times, after digesting some each time, extract the supernatant with a pipette, and centrifuge the result that is the cardiomyocytes. Taking advantage of the difference in wall-application time between cardiomyocytes and fibroblasts, differential wall-application was used for 1.5 h to remove fibroblasts sufficiently and achieve the purpose of purification. Fibroblasts have a pike-shaped or irregularly triangular cytosol with an ovoid nucleus in the center, protruding cytoplasm, radial growth, and proliferation without pulsation, which is good to distinguish from cardiomyocytes. Digestion and blowing must not be excessive, is the most important principle to ensure the viability of cardiomyocytes. The inoculation density (generally not less than 105/ml ) will also affect the beating and synchronized beating of cardiomyocytes. For more product details, please visit Aladdin Scientific website.
Low sugar DMEM, trypsin EDTA penicillin Sodium bicarbonate solution Neonatal bovine serum Glutamine D-Hank's liquid Neosporin Iodine Alcohol
Aluminum box Ophthalmic straight scissors Ophthalmic curved scissors Ophthalmic straight forceps Ophthalmic curved scissors Glass Petri dishes Wide-mouth flasks Cotton balls Beakers Cone flasks Centrifuge tubes Magnetic stirrer Stirrer Water bath oscillator Nylon sieves Needle filters
1. Animal
15 SD suckling mice 1-4 days after birth.2. Reagents
Low-sugar DMEM, trypsin (containing EDTA), penicillin, sodium bicarbonate solution, neonatal bovine serum, glutamine, D-Hank's solution, 0.1% Neosporin, iodine, 75% alcohol, culture medium (DMEM, neonatal bovine serum, penicillin).3. Surgical instruments and apparatus
Aluminum box, ophthalmic straight scissors, ophthalmic curved scissors, ophthalmic straight forceps, ophthalmic curved scissors, glass Petri dishes (2 sets, one set for dissection and the other set for shearing cardiac tissues), two 100 ml wide-mouth flasks (for iodine and alcohol cotton balls, respectively), 500 ml beakers, 250 ml conical flasks, centrifugal tubes of 15 ml and 50 ml, magnetic stirrers and stirrers (or water bath oscillators), 150-200 mesh nylon sieves and needle filters.Methods
1. Trypsin was prepared to a concentration of 0.06% with D-Hank's liquid and incubated at 37°C in a water bath.2. Anatomy: Soak the suckling mouse in 0.1% Neosporin and take it out. Use another pair of large forceps to take iodine cotton balls to rub the skin, and then use alcohol cotton balls to remove iodine. The skin on the back of the neck was pinched with the left hand to fully expose the chest. With the right hand, a pair of ophthalmic straight scissors was used to cut open the skin and fully tear it apart, and then after sterilizing the skin with alcohol, a pair of ophthalmic curved scissors was used to cut open the ribs upward along the left lower edge of the sternal stem, and then the sternum was cut horizontally in the middle of the incision. In this way, the heart of the suckling mouse jumped out directly with a slight top of the left hand. The ventricular portion of the heart is then cut off directly from the middle of the heart with curved ophthalmic forceps and placed in an ice bath of D-Hank's solution. The process was repeated. Once the removal is complete, the surgical instruments for removal are removed.
Note: In order to ensure the viability of the cardiomyocytes, it is important to remove the heart as quickly as possible. In addition, it is best to place the heart in a petri dish on an ice bench or in pre-cooled, balanced saline solution.3. Perform the following operations with a second set of surgical instruments. Use ophthalmic straight forceps and ophthalmic curved scissors to remove blood clots and fibrous tissue from the periphery of the heart in the Petri dish and place it in another Petri dish pre-filled with D-Hank's solution. After washing the heart tissue again, place the heart tissue in another Petri dish (or other suitable container depending on personal preference), add a small amount of 0.06% pancreatic enzyme, and use ophthalmic curved scissors to cut the heart tissue into pieces of 1 mm3 in size. The heart tissue should be cut into 1mm3 pieces with ophthalmic curved scissors, and the cut heart and trypsin solution should be transferred into a conical flask with a stirrer, and 2-3 ml of fresh trypsin should be sucked up to rinse the dish and the scissors and transferred into the conical flask, and the trypsin should be added to the final volume of 10 ml, and then put into a water bath of 37℃ (a sterilized 500 ml beaker can be used to fill with sterile distilled water, and the volume of water added is enough, and the water level should be slightly lower than the scale of the 250-ml conical flask, too little will cause the temperature to rise. line is sufficient, too little will result in uneven temperature and too much will be easily contaminated. Turn on the power of the magnetic stirrer, adjust the water temperature to 37°C beforehand), adjust the speed to about 60 rpm, and digest for 15 min (make sure the temperature of the water bath is constant at 37°C, and the digestion time is not more than 15 min). Alternatively, if a water bath shaker is used, it is not necessary to add a stirrer, and the digestion can be done directly in the water bath shaker, with the same rotation speed and digestion time.4. Remove the conical flask from the water bath and carefully aspirate the supernatant, which consists mainly of red blood cells and fibroblasts.5. Add 10 ml of new trypsin and blow the solution several times with a new pipette to mechanically disperse the cells (the tissues will become viscous and colloidal after digestion), note: do not blow too much or it will lead to over-digestion of the tissues, and then digest for 10-15 min at 37℃ with stirring; if the number of suckling mice is small (e.g., when there are 5 or 6 mice), the digesting time can be reduced to 5-10 min or else it is easy to over-digest. If the number of mice is small (e.g. 5 or 6 mice), the digestion time can be reduced to 5-10 min, otherwise it is easy to over-digest. At the same time of digestion, add 20 ml of pre-cooled culture medium containing 10% serum to a 50 ml sterile disposable centrifuge tube and place it on the ice. After digestion, carefully transfer the supernatant to the centrifuge tube with culture medium, and collect the isolated cells for the first digestion. After the first digestion, continue with the second digestion, and add 10 ml of fresh trypsin to the remaining tissue mass for further digestion.6. Repeat the 5 digestion steps until a small amount of tissue mass remains, generally 4-5 times can digest most of the tissue mass (excluding the time when the digested liquid is discarded).7. The first and second cell-containing digests are placed in a centrifuge tube, passed through a 180-mesh screen, and centrifuged together; the third and fourth cell-containing digests are placed in a centrifuge tube, passed through a 180-mesh screen, and centrifuged together. Finally, the two tubes of cells were resuspended with 8 ml of culture medium containing 20% serum, inoculated into a 75 cm2 plastic culture bottle, placed in an incubator for 1.5 hours, then removed, discarded the adherent cells (mainly fibroblasts and endothelial cells), and took out the non-adherent cells, counted them by Tepan Basket Reject Dyeing Method, and then adjusted the concentration of the cells to 5-6 x 105 cells/ml by adding the culture medium and inoculated into the intended culture vessel. The cell suspension was taken out, counted by Tepan basket rejection method, added culture medium to adjust the cell concentration to 5-6x105 cells/ml, and inoculated into the target culture vessel.5. Generally there is no need to add BrdU, if the culture time is long (and very few fibroblasts are found), 0.1 mmol/ml BrdU (Sigma) can be added to prevent the proliferation of fibroblasts. With the addition of BrdU, the duration of cardiomyocyte beats will be longer.9. 24 hours after inoculation, gently rinse the cultured cardiomyocytes once with warm medium or balanced saline solution to remove the unaffixed cells (some of the cells will be affixed after 24 hours, resulting in overlapping growth of many cells), and then replace the culture medium. Treatment factors can be applied after 48 h or 72 h of incubation, depending on the needs of the experiment.Results
Cardiomyocytes were round or oval in shape when they were first inoculated, and basically adhered to the wall around 24 h. At this time, the cells stretched out pseudopods, which were fibrous strips under the microscope, and the cell bodies showed irregular shapes, such as polygonal, and some of the cells had the tendency of aggregation, and the cells had a better pulsation effect, and the DMEM medium was dark red at the initial incubation, and then turned into yellowish after two days, which should be the performance of the decline in nutrient content, and also indicated the good growth condition of cells. It should be a sign of decreasing nutrient content, and also indicates that the cell growth condition is good. We also refer to some foreign literature for culture methods to supplement. The easiest way to identify the cells is whether they are pulsating or not, note: when the pulsation is weak, you may not see the pulsation under 10x objective lens, but you may be able to observe it under 20x or 40x objective lens. The best way to test the operation is to look at the purity of the cardiomyocytes and their ability to beat. In addition, cardiomyocytes express actin, which can be used as an identifier but is not the only characterization, because smooth muscle cells also express it.
The shape of cardiomyocytes was round or oval when they were first inoculated, and they were basically attached to the wall around 24 h. At this time, the cells stretched out the pseudopods, which were fibrous strips under the microscope, and the cell bodies showed irregular shapes, such as polygonal, and some of the cells had the tendency of aggregation, and the cells had a better pulsation effect, and the DMEM medium was deep red at the initial culture, and turned into yellowish after two days, which should be the manifestation of the decline of nutrient content, and also indicated the good growth condition of cells. It should be a sign of decreasing nutrient content, and also indicates that the cell growth condition is good. We also refer to some foreign literature for culture methods to supplement. The easiest way to identify the cells is whether they are pulsating or not, note: when the pulsation is weak, you may not see the pulsation under 10x objective lens, but you may be able to observe it under 20x or 40x objective lens. The best way to test the operation is to look at the purity of the cardiomyocytes and their ability to beat. In addition, cardiomyocytes express actin, which can be used as an identifier but is not the only characterization, because smooth muscle cells also express it.